1. Field of the Invention
Lipases (glycerol ester hydrolases EC 3.1.1.3) are enzymes which hydrolyze the glycerol esters of long chain fatty acids (glycerides).
This invention relates to the production of a lipase from Rhizopus delemar (R. delemar) using recombinant DNA (deoxyribonucleic acid) methods. The lipase coding gene from R. delemar is inserted into host microorganisms for the production of lipase products.
The uses of lipases to exchange glyceride fatty acids with free fatty acids or those of other glycerides and for the conduct of a range of chemical reactions in organic solvents have been described. Lipases can also be used to release fatty acids from glycerides.
Commercially, fatty acids are important chemicals used in a variety of consumer goods including cosmetics, foods, plastics, paints, varnishes, lubricating greases and household cleaners. The major sources of fatty acids are fats and oils from animals and plants. Fats and oils are composed of triglycerides, that can be subsequently broken down to free fatty acids and glycerol. Free fatty acids have been traditionally prepared by chemical hydrolysis using both high temperature and pressure. An alternative to chemical hydrolysis is an enzymatic process using lipases.
Enzymatic hydrolysis of triglycerides has the advantage of a reaction environment at reduced temperature and pressure. Lipase, although overcoming the disadvantages of chemical hydrolysis, does not completely split the triglyceride molecule. The disadvantage, however, of this enzymatic hydrolysis is that it necessitates use of considerable amounts of a very expensive enzyme. As such, there is a need for the production of an inexpensive lipase available in quantity.
Molecular cloning and expression of a lipase gene offers a method to address this need. In addition, the availability of a cloned lipase gene facilitates the use of site directed mutagenic techniques to modify lipase so as to improve its utility.
2. Description of the Prior Art
Lipase cDNA has been cloned and sequenced from a similar fungus Rhizomucor miehei. Sequenced inserts from E. coli yielded a probable amino acid sequence of the extracellular lipase secreted by that organism. While sequence information was obtained there was no expression by the transformed host, Boel, et. al., Lipids, Vol. 23, No. 7, (1988), p. 701. The sequence of the cDNA from R. miehei is not identical to that reported here.
Lipases from R. delemar have been purified to varying degrees, Chiba, et al., Biochem. Biophys. Acta, Vol. 327, (1973), p. 380; Fukumoto, et al., J. Gen. Appl. Microbiol., Vol. 10, No. 3, (1964), p. 257; Iwai and Tsujisaka, Agr. Biol. Chem., Vol. 38, (1974), p. 1241; Mohsen, et al., Egypt. J. Food Sci., Vol. 14, No. 1, (1986), p. 147. In none of these reports is the lipase demonstrated to be free of contaminating non-lipase proteins.
It has recently been determined that a partially purified sample of crude R. delemar lipase contained at least 10 different proteins, Antonian, Lipids, Vol. 23, No. 12, (1988), p. 1101. Due to the discrepancies in the literature, it is apparent that prior to the instant invention, no single lipase from R. delemar has been purified to homogeneity.
Kouker and Jaeger [Appl. Environ. Microbiol., Vol. 51, (1987), p. 211] have described a method employing medias containing Rhodamine B and olive oil for the detection of naturally occurring organisms which produce lipase. However, no such method has been described for the screening of organisms containing recombinant DNAs.